MicroRNAs (miRNAs) have been increasingly recognized to play important roles in normal physiology and in pathological processes, including cancer. We recently identified the miR-200 family and the miR-194/192 cluster of miRNAs as negative regulators of epithelial-mesenchymal transition (EMT), which is thought to be the critical initiating step of cancer metastasis. These miRNAs maintain the epithelial cell phenotype and inhibit EMT by repressing the expression of transcriptional inhibitors of E-cadherin, including ZEB1/ZEB2 and MeCP2. Overexpression of these miRNAs inhibits TGF-induced EMT in normal mammary epithelial cells. Furthermore, ectopic expression of the EMT-related miRNAs in invasive breast tumor cells induces mesenchymal-epithelial transition (MET), the reverse process of EMT, with significant reduction of migration and invasion. Surprisingly, we also found that elevated miR-200 expression is correlated with increased ability of tumor cells to generate macroscopic lesions at secondary metastasis sites. These data in aggregate imply that there is a stage-specific role of these miRNAs in metastasis. Decreased miRNA expression in the primary tumor may stimulate EMT and subsequent dissemination of tumor cells, whereas increased miRNA expression will lead to MET and colonization of distant organs. As potentially new agents for anti-metastasis therapeutics, the functional role and molecular mechanism of these miRNAs in different stages of cancer progression require further characterization. We hypothesize that these miRNAs are critical master regulators of the transition between the epithelial and mesenchymal states of tumor cells and play important roles in both the initial invasion of primary tumors as well as metastatic colonization of secondary target organs. We will use a comprehensive approach to study the molecular mechanism of miRNA-mediated EMT and cancer metastasis, taking advantage of in vitro and in vivo models as well as genomic and proteomic research platforms available in our laboratory. To study the potential biphasic role of these miRNAs in metastasis, we will employ orthotopic and experimental metastasis assays. We will overexpress the miRNAs and modulate their downstream genes of interest in various mouse and human breast cancer cell lines and utilize in vivo animal models to investigate stage-specific effects on breast cancer metastasis (Aim 2). We will use combined analysis of gene expression profiling and mass spectrometry to identify novel target genes of EMT-related miRNAs and test the functional importance of these genes in EMT and metastasis using well-established in vitro and in vivo assays (Aim 2). Finally, we will evaluate the importance of EMT-related miRNAs in mammary gland function and mammary tumor progression using transgenic mouse models. We will generate MMTV transgenic mice for these miRNAs and test the role of these miRNAs in breast cancer progression in tumor-prone animals that overexpress the oncogene Neu or PyMT in their mammary tissue (Aim 3). Through these experiments, we will reveal the potential stage-specific roles and functional mechanisms for EMT-related miRNAs in cancer metastasis and provide important novel insights for improving the prevention and treatment of metastatic cancer. PUBLIC HEALTH RELEVANCE: Over 90% of cancer related deaths are due to the metastatic spread of primary tumor cells to distant vital organs. Epithelial-mesenchymal transition (EMT) and the reverse process, MET, have been postulated to be important processes for tumor cells to gain the ability to migrate and invade into their surrounding tissue and to regain their epithelial phenotype to facilitate colonization once they reach the target organs. This study will investigate how microRNAs, a novel class of genetic regulators, regulate EMT and will provide some of the best evidence to date as to whether EMT can promote early dissemination of tumor cells while MET may be essential for efficient colonization of distant target organs.